Purification Chemistries and Formats for Sanger DNA Sequencing Reactions on a Micro-Fluidics Device

ABSTRACT

According to various embodiments described herein, a microfiuidics-chip based purification device and system for Sanger-sequencing reactions is provided. The device and system allow for the introduction into a sequencing system of a cartridge containing purification technologies specific to the sequencing contaminants or sequencing method where the simplified purification solution of a cartridge allows automation of the sample purification process, reduced consumption of purification reagents, and consistency in sampling by reducing the sampling errors and artifacts. These various embodiments therefore solve the need for a microfiuidics-chip-based, Sanger-sequencing reaction purification system for CE devices. The microfiuidic chips described can be used as a PCR chip by reorganizing the on-chip reagents, reaction wells and work flow steps.

BACKGROUND OF THE INVENTION

DNA sequencing is the process of determining the precise order ofnucleotides within a DNA molecule. Two common sequencing methods areSanger sequencing and “Next-Gen” sequencing, where Sanger sequencing isa method of DNA sequencing based on the selective incorporation ofchain-terminating dideoxynucleotides (or ddNTPs) by DNA polymeraseduring in vitro DNA replication. While “Next-Gen” sequencing methods aretypically used for large-scale, automated genome analyses, Sangersequencing is primarily used for smaller-scale projects with the goal ofobtaining especially long contiguous DNA sequence reads (>500nucleotides). The use of labeled (either radioactively or fluorescently)amplification compounds (modified nucleotides or ddNTPs) for detectionin automated sequencing machines typically results in contamination ofsubsequent sequences if the systems are not properly decontaminated andcleaned. Because avoidance of any overlap in amplification compounddetection requires precise adjustment of all amplification products, thecleanup of, for example, fluorescence-based Sanger sequencing reactionsis a crucial sample preparation step before subsequent sample analysis.In the context of other DNA purification methods, sequencing reactionpurification or cleanup efforts generally focus on single-stranded DNAand generally requires expulsion to a high degree of the sequencingreaction contaminants, including buffering salts (de-salting), otherions, and unincorporated dye-ddNTP (dye-terminator) such that ionicstrength is significantly reduced relative to the original reaction. Forexample, a target for residual salt level after purification could beless than or equal to 5 mM. Further, for dye terminators, apost-purification target could be greater than 1000-fold reduction fromthe original dye terminator concentration.

As a result, what is needed is a system to automate and simplify thegeneration of a clean sequencing sample that, except for the potentialaddition of certain compounds (such as formamide) and certain processes(such as heat-denaturation), yields a sequencing solution with lowcontaminant concentrations and is ready for electrokinetic injection andelectrophoretic separation by capillary electrophoresis (CE).

A further need, for reasons discussed below, is the implementation ofsuch a system for purification on a microfluidics chip, as known methodsfor purification have not been implemented on a microfluidics chip.

Purification of double-stranded DNA on microfluidics chips (for example,during the extraction and purification of biological samples such aswhole blood or the clean up of PCR-products) have been largely describedin the literature. However, only a limited amount of research has beendevoted to systems allowing on-chip purification of Sanger sequencingsingle-stranded DNA reactions because sequencing is a very demandingapplication to integrate on a microchip, by requiring two rounds ofthermocycling, with each requiring subsequent cleanup. Most publishedDNA assay chips (e.g., Rheonix Card®) are more modest in scope,implementing simpler workflows, or performing simple 1- or 2-stepprotocols like the gDNA preparation or one thermocycling PCR reactionalone.

One of the few publications demonstrating a microfluidics-based Sangersequencing reaction cleanup (Mathies group of Blazej, Kumaresan andMathies) describes an affinity capture/electro-elution chipfunctionality for the purpose of sequencing reaction clean-up. Inparticular, universal capture oligonucleotides covalently attached tothe surface of a gel matrix ‘capture gel’ (created in one area of thechip) hybridize the sequencing products. Although this system allows forremoval of charged impurities such as excess dye-terminator and salt byelectrophoretic-elution, the sequencing reaction cleanup method requiresthe chip to be electrically connected and able to performelectrophoresis to function.

SUMMARY OF THE INVENTION

According to various embodiments described herein, a microfluidics-chipbased purification device and system for Sanger-sequencing reactions isprovided. The device and system allow for the introduction into asequencing system of a cartridge containing purification technologiesspecific to the sequencing contaminants or sequencing method where thesimplified purification solution of a cartridge allows automation of thesample purification process, reduced consumption of purificationreagents, and consistency in sampling by reducing the sampling errorsand artifacts. These various embodiments therefore solve the need for amicrofluidics-chip-based, Sanger-sequencing reaction purification systemfor CE devices. Though the following focuses on Sanger-sequencingreaction purification systems for CE, the microfluidic chips describedcan be used as a PCR chip by reorganizing the on-chip reagents, reactionwells and work flow steps.

In an embodiment, a microfluidic sequencing reaction purification deviceis provided for reducing the number of sequencing contaminants in asingle-stranded DNA sequencing sample. The microfluidics chip-basedsequencing reaction purification device can comprising a surface, asolid-phase extraction substrate and silane bound to a structure. Thestructure can be selected from the group consisting of microstructures,the surface of the microfluidic device, a membrane, a high-surface area,convoluted material, and combinations thereof.

In another embodiment, a microfluidic sequencing reaction purificationdevice is provided for reducing the number of sequencing contaminants ina single-stranded DNA sequencing sample. The microfluidic sequencingreaction purification device can comprise a surface and a reagent boundto a structure. The structure can be selected from the group consistingof microstructures, the surface of the microfluidic device, a membrane,a high-surface area, convoluted material, and combinations thereof.

In a further embodiment, a microfluidic sequencing reaction purificationsystem is provided for reducing the number of sequencing contaminants ina single-stranded DNA sequencing sample and automating the purificationprocess. The microfluidics chip-based sequencing reaction purificationsystem can comprise a DNA sequencing system, a microfluidics deviceconfigured to operate within the DNA sequencing system. Themicrofluidics chip can comprise a surface and a reagent bound to astructure selected from the group consisting of a solid-phase extractionstructure, microstructures, the surface of the microfluidics device, amembrane, a high-surface area, convoluted material, and combinationsthereof.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates sequencing data from sequence purified usingChargeSwitch® in 96 well plate.

FIG. 2 illustrates sequencing data from sequence purified usingDynabeads® in 96 well plate.

FIG. 3 illustrates a side view of a flow-channel according to oneembodiment of the invention.

DETAILED DESCRIPTION

The following description provides embodiments of the present invention.Such description is not intended to limit the scope of the presentinvention, but merely to provide a description of embodiments.

Several formats are possible for the implementation of purificationmedia on a microfluidics device. Formats include, for example,micro-beads (for example polystyrene, latex beads or ion-exchangeresin), modified surface on the microfluidics device, frits andmembranes composed of DNA binding material and paramagnetic beads.

Combinations of non-limiting examples of purification methods andformats are summarized in the following table. Selection can be based oncommercial availability, ease of implementation on a microfluidicsdevice and least required R&D development effort.

Surface coating (Non- Para- of the magnetic) magnetic micro Frit ormicro micro fluidics wool Chemistry/Format beads beads device Membranematerial Silane X ChargeSwitch ® X X BDX ® X PureLink ® X HybridizationX based pull-out Size Exclusion X

Each of these exemplary formats are discussed below in relation toexemplary purifications chemistries.

1. Purification by Solid Phase Extraction Utilizing Silane

Solid-phase extraction (SPE) is a separation process by which compounds(solutes) dissolved or suspended in a liquid mixture are separated fromother compounds in the mixture according to their physical and chemicalproperties by using the affinity of the solutes for a solid throughwhich the sample is passed to separate the mixture into distinctcomponents. In one embodiment, the microfluidics-chip-based purificationdevice is a solid phase extraction cartridge utilizing silane bound tomicrobeads, membranes, or microstructures within the device (e.g.,plastic tubes or plates). In one embodiment, the microfluidics deviceand silane-coated microbeads is utilized in combination as a Sangersequencing reaction purification system. In alternative embodiments, thesilane-coated microbreads may comprise polystyrene, latex, agarose, anion-exchange resin, an immobilized metal affinity chromatography (IMAC)resin, or any other substrate or resin capable of being coated by silaneand utilized in a Sanger sequencing reaction purification system. In yetanother embodiment of the purification system utilizing silane-coatedmicrostructures (such as microbeads, tubes, plates, or any combinationof these structures), the microstructures may be non-magnetic, magnetic,or paramagnetic. In yet another embodiment, the silane is bound to astructure including, but not limited to, a frit, a wool, a membrane orany other high-surface area, convoluted material, structure, or compoundcapable of being incorporated into a microfluidics device. Themicrofluidics device can be, for example, a microfluidic chip, card orcartridge.

In an embodiment, silane-coated microbeads (e.g., paramagnetic beads)bind single stranded DNA (ssDNA) and RNA. DNA can be bound in very lowcopy numbers. For example, as little as 10 copies of M13 single-strandedDNA could be captured and eluted from the paramagnetic beads. In atypical silane-coated bead purification protocol, 2 mg are used in a 400μl bind reaction volume to capture approximately 5 μg of genomic doublestranded DNA onto the surface of the paramagnetic micro beads. Theconditions chosen for these experiments were such that the standard(tube)-scale amounts of beads (2 mg per assay) were used to extract thessDNA in a 400 μl volume. Moreover, since the beads are paramagnetic,they can be easily immobilized in a microfluidic device (e.g.,cartridge) format.

2. Purification by Reversible Ion-Exchange Binding of DNA

Reversible ion-exchange binding of DNA is a purification, separation, ordecontamination process by which ions are exchanged between twoelectrolytes or between an electrolyte solution and a complex. Theprocess typically involves solid polymeric or mineralic ion exchangers(e.g., ion exchange resins (such as functionalized porous or gelpolymers), zeolites, and montmorillonite, clay, or soil humus). Ionexchangers can also include, for example, ionizable (or switchable) ionexchangers. In an embodiment of an ion-exchanger, the ion-exchangercomprises a surface ligand whose surface charge is a function of pH. Theion-exchanger surface ligand, for example, can be positively charged atlow pH, and neutral at pH 8.5, to bind and elute plasmid.

In an alternative embodiment of the purification system, a microfluidicsdevice comprising microstructures (such as microbeads, tubes, or plates)coated in at least one ion-exchanger is utilized as a Sanger sequencingreaction purification system. For example, the surface of themicrofluidics device can be coated in at least one ion-exchanger. Inanother example, the microfluidics device can comprise a membrane coatedwith at least one ion-exchanger. In yet another example, theion-exchanger can be bound to a structure including, but not limited to,a frit, a wool, a membrane or any other high-surface area, convolutedmaterial, structure, or compound capable of being incorporated into amicrofluidics device. In a further example, one or any combination ofthe microstructures, the device surface, a high-surface area structure,or a membrane can be coated in an ion-exchanger as described above. Inanother embodiment of the purification system utilizingion-exchanger-coated microstructures (such as microbeads, tubes, plates,or any combination of these structures), the microstructures may be, forexample, non-magnetic, magnetic, or paramagnetic. The microfluidicsdevice can be, for example, a microfluidic chip, card or cartridge.

3. Purification by Size Exclusions and Ion-Exchange (SEW)

Size exclusion and ion-exchange (SEIE) is a process in which moleculesin solution are separated by their size (e.g., molecular weight) andcharge. In an alternative embodiment of the purification system, aSanger sequencing reaction purification system utilizes a microfluidicsdevice and reagents specifically chosen to sequester reaction componentsbased on the components charge and size. These reagents can be utilizedby the microfluidics device to capture unincorporated dye exterminators,dNTPs, free salts, or salt ions generated during the sequencingreaction. The reagents can be bound to microstructures, where themicrostructures may comprise microbeads, membranes, or structures withinthe chip (e.g., plastic tubes or plates). The reagents can be bound tomicrostructures (such as microbeads, tubes, plates, or any combinationof these structures) that may be, for example, non-magnetic, magnetic,or paramagnetic. Alternatively, the reagents can be bound to the surfaceof the microfluidics device, or can also be bound to a structureincluding, but not limited to, a frit, a wool, a membrane or any otherhigh-surface area, convoluted material, structure, or compound capableof being incorporated into a microfluidics device. Finally, thesize-exclusion and ion-exchange reagents can be bound to one or anycombination of microstructures, the surface of the microfluidics device,or a high-surface area, convoluted structure within the microfluidicsdevice as described above. The microfluidics device can be, for example,a microfluidic chip, card or cartridge.

In contrast to other cleanup chemistry (bind-wash-elute style), sizeexclusion beads work by binding the known impurities. Sephadex® beads,for example are very cost-effective commercially available sizeexclusion beads that have the ability to extract terminators and saltwhile leaving the products of the sequencing reaction in solution. Thesebeads advantageously have a relatively low cost. Moreover, they havebeen used routinely for sequencing reaction cleanup in conjunction with96-well filter plates (for example, MultiScreen® 96w plates (Durapore®or Ultracell®-10 filter) from Millipore).

4. Purification by Membrane

Purification by membrane is a mechanical separation process in whichundesirable sequencing solution reaction compounds are removed from thesystem using binding buffers and a porous physical structure (such as amembrane) through which the bound reaction compounds cannot pass. In oneembodiment, the system uses at least one binding buffer and at least oneporous structure configured to remove reaction compounds such as shortprimers, dNTPs, enzymes, short-failed PCR/CE products, salts from PCR/CEproducts, or any combination thereof. In an alternative embodiment ofthe purification system, a Sanger sequencing reaction purificationsystem utilizes a microfluidics device comprising at least one bindingbuffer and at least one membrane. The membrane may be any structureincluding, but not limited to, a frit, a wool, a membrane, or anyporous, high-surface structure through which a solution may pass. Themicrofluidics device can be, for example, a microfluidic chip, card orcartridge. An example of purification by membrane is PureLink®,manufactured by Life Technologies.

5. Purification by Hybridization-Based Pull-Out

Hybridization-based pull-out is based on hybridization-binding of thesequencing reaction products to a complementary oligonucleotide that isattached or bound to a microstructure or the surface of themicrofluidics device. In an embodiment of the purification system, aSanger sequencing reaction purification system utilizes a microfluidicsdevice comprising at least one hybridization-based pull-outoligonucleotide selected to be complementary to a sequencing reactionproduct. In an alternative embodiment, the hybridization-based pull-outoligonucleotide is bound to a microstructure or the surface of themicrofluidics device. In another embodiment of the purification systemutilizing hybridization-based pull-out oligonucleotides bound tomicrostructures (such as microbeads, tubes, plates, or any combinationof these structures), the microstructures may be, for example,non-magnetic, magnetic, or paramagnetic. The hybridization-basedpull-out oligonucleotide can be bound to a structure including, but notlimited to, a frit, a wool, a membrane or any other high-surface area,convoluted material, structure, or compound capable of beingincorporated into a microfluidics device. The hybridization-basedpull-out oligonucleotide can also be bound to one or any combination ofmicrostructures, the surface of the microfluidics device, or ahigh-surface-area, convoluted structure as described above. Themicrofluidics device can be, for example, a microfluidic chip, card orcartridge.

Hybridization beads, for example, with capturing oligonucleotideattached to its surface, can be made by numerous manufacturing processesincluding, for example, the process used to manufacture Anti-miRNA BeadCapture (ABC) beads. The ABC kit is a commercialized product forcapturing specific miRNA from a biological sample (e.g., blood)directly. It can use a complementary oligonucleotide on a magnetic beadto hybridized and capture specific miRNA. The bead-oligo linkage iscovalent and permanent using the well-known carboxy (on bead), NH-ester(on oligo) standard chemistry.

6. Purification Combining Multiple Purification Methods

In another embodiment, the microfluidics device comprises multipletechnologies, including, but not limited to, solid-phase extractiontechnology utilizing silane, reversible ion exchange binding of DNA,size exclusion and ion-exchange technology, membrane technology, andhybridization-based pull-out technology.

Examples

ChargeSwitch® (product of Life Technologies) Purification example ofreversible IE binding of DNA:

Polymer Preparation:

Bis-Tris is reacted with Polyacrylic Acid in the presence of EDC toyield a polymer containing bound Bis-Tris.

-   -   Polymer bound Bis-Tris can be protonated by acid (H+) in acidic        conditions producing a positive charged surface. The positively        charged polymer selectively binds to DNA.    -   At higher pH (>8), the polymer should still be water soluble and        have no charge; effectively enabling elution of bound DNA

Protocol to Coat Polymer on Solid Surface:

-   -   1. Polymer can be diluted in 1% PB buffer.    -   2. 100 μl of 10-100% polymer is added to the solid surface to be        coated.    -   3. Wait 15 min and discard polymer from the surface    -   4. Add 100 μl of 1% PB buffer, wait 2 min and remove the buffer    -   5. Step 4 is repeated again    -   6. Air dry solid surface for at least 2 hours

Protocol to Purify Sequencing Reactions

a. With polymer coated on solid surface.

-   -   1. Add 10 μl of sample to the coated surface.    -   2. Bind the sample by adding and equivalent volume (10 μl) of        DCB to it.    -   3. Incubate for 15 min, remove the liquid completely.    -   4. Wash the sample by adding 150 μl of DCBW to the plate        location    -   5. Incubate for 1 min, remove the liquid completely    -   6. A final wash of sample is done by flowing 150-200 μl of        nuclease-free-water over the surface        -   (If possible, minimize the time from adding the water to            removing it in step 11 as much as possible to prevent            possible elution of the bound sample.)    -   7. Elute the sample by adding 10 μl of HDF or Tris HCl (pH 8.5)    -   8. Incubate for 5 min and then collect the supernatant

b. With polymer coated on magnetic beads.

-   -   1. To 10 μl of sample, add 10 μl DCB42 buffer and 2 μl of beads        (stable at RT)    -   2. Mix and incubate at RT for 7 min    -   3. Magnetize beads and discard supernatant    -   4. Demagnetize and add 150 μl of DCBW6 wash buffer and mix    -   5. Magnetize beads and discard supernatant    -   6. Repeat step 4 & 5    -   7. Demagnetize and add 10 μl of elution buffer    -   8. Mix and incubate for 5 min    -   9. Magnetize beads and collect supernatant to sample outlet

FIG. 1 illustrates sequencing data from sequence purified usingChargeSwitch® in 96 well plate.

DynaBeads® Purification example of SPE:

Bead Preparation:

Dynabeads® MyOne™ SILANE (product of Life Technologies) are supplied ata concentration of 40 mg/ml. Prior to use, the beads should betransferred to the appropriate binding solution as follows:

-   -   1. Re-suspend the Dynabeads® MyOne™ SILANE completely (e.g.        vortex) to a homogenous suspension prior to use. Leave on a        roller until use.    -   2. Transfer 400 μl of re-suspended Dynabeads® MyOne™ SILANE to a        fresh tube. Place the tube on the magnet until the supernatant        is clear, then remove and discard the supernatant.    -   3. Re-suspend in 13.33 μl of 40% TEG (tetraethlyene glycol)    -   4. Add 240 μl 100% ethanol and mix thoroughly.    -   5. The final bead-solution used in the isolation protocol        described below should contain Dynabeads® MyOne™ SILANE at 1.5        mg/ml in 2% TEG/90% ethanol.

Protocol for Sequence Clean-Up

-   -   6. Add 20 μl (30 μg) Dynabeads® MyOne™ SILANE (supplied in TEG        and ethanol, see above) to 10 μl sequencing reaction mix.    -   7. Mix and incubate for 10 minutes at room temperature.    -   8. Magnetize the beads and remove the supernatant completely.    -   9. Demagnetize and re-suspend the Dynabead® MyOne™ SILANE in 30        μl 55% ethanol.    -   10. Magnetize the beads and remove the supernatant completely.    -   11. While still on the magnet, let the Dynabeads® MyOne™ SILANE        pellet air-dry for 5 minutes at room temperature.    -   12. Demagnetize and re-suspend the Dynabeads® MyOne™ SILANE in        10 μl water.    -   13. Incubate for 3 minutes at room temperature.    -   14. Magnetize beads again and transfer the supernatant        containing the sequencing products for sequencing readout.

FIG. 2 illustrates sequencing data from sequence purified usingDynabeads® in 96 well plate.

BigDye XTerminator® example of SEIE purification. BigDye XTerminator®(BDX) (product of Life Technologies) is used for sequencing purificationin 96 or 384 well-based plates. BDX beads capture the left over wasteproducts from the BDX reactions. The BDX beads are two bead types. Thefirst is an ion exchange bead designed to capture negative chargeditems. These beads are also coated with a surface that will preventlarge negative charged sample fragments from binding. The secondbead-type is an ion exchange bead that captures positive chargedmoieties.

Procedure to Purify Sequencing Product on Chip Using BDX

-   -   1. Add 45 μl of SAM solution and 5 μl of beads solution to 10 μl        of sample at RT    -   2. Mix the liquid (by moving back and forth or as appropriate)        for 20 min    -   3. The supernatant containing the sequencing reaction can be        separated from beads using various methods:        -   a. Magnetization can be used if BDX beads are coated on            magnetic core        -   b. Micro pore filters can be used to retain the beads.        -   c. A flow-channel 10 can be designed to retain beads when            liquid passes through it, as illustrated in FIG. 3. Inlet 20            accepts a mix of beads and liquid with trapped beads 30            allowing clear liquid 40 to pass through the channel.            -   a. The beads can be staged such that the first beads                capture the positive charged moieties and the second                stage captures the negative charged moieties. The BDX                beads also have a size exclusion coating that prevents                the longer DNA sample fragments from being captured. The                beads can be staged so that the sample does not become                clogged. This may require multiple stages of beads.        -   d. Mixing can be used to promote efficient waste capture in            the beads. Magnetic beads can aid mixing. The beads can            transit between multiple chambers or regions in single            chamber with magnets that oscillate between positions.        -   e. If beads are captured in a frit then the sample can            oscillate back and forth in the beads. Alternatively, a            fluid path could be created where the sample is circulated            through bead region. After a given number of passes, a valve            is opened and the clean sample is allowed to pass to the            next stage.        -   f. The beads and sample can be heated to increase the            reaction rate of the waste to the beads. Heating can also            help when charge switch or silane beads are used.        -   g. Membranes with the same ion exchange and size exclusion            properties can replace the BDX beads. One membrane would            capture negative ions and a second the positive ions. Mixing            would be achieved as for beads in a frit.    -   4. Collect the liquid for sequencing readout.

The preceding descriptions of various implementations of the presentteachings have been presented for purposes of illustration anddescription. It is not exhaustive and does not limit the presentteachings to the precise form disclosed. Modifications and variationsare possible in light of the above teachings or may be acquired frompracticing of the present teachings. Additionally, the describedimplementation includes software but the present teachings may beimplemented as a combination of hardware and software or in hardwarealone. The present teachings may be implemented with bothobject-oriented and non-object-oriented programming systems.

1. A microfluidic sequencing reaction purification device for reducingthe number of sequencing contaminants in a single-stranded DNAsequencing sample, the microfluidics chip-based sequencing reactionpurification device comprising, a surface; a solid-phase extractionsubstrate; and silane bound to a structure selected from the groupconsisting of microstructures, the surface of the microfluidic device, amembrane, a high-surface area, convoluted material, and combinationsthereof.
 2. The microfluidic sequencing reaction purification device ofclaim 1, wherein the microstructure comprises microbeads, tubes, orplates.
 3. The microfluidic sequencing reaction purification device ofclaim 1, wherein the microstructures comprise polystyrene, latex,agarose, an ion-exchange resin, an immobilized metal affinitychromatography resin, or any other substrate or resin capable of beingcoated by or bound to silane.
 4. The microfluidic sequencing reactionpurification device of claim 2, wherein the microstructures arenonmagnetic, magnetic, or paramagnetic.
 5. The microfluidic sequencingreaction purification device of claim 1, wherein the high-surface area,convoluted material comprises frit or wool.
 6. The microfluidicsequencing reaction purification device of claim 1, wherein thesequencing contaminants comprise salts, free salts, salt ions, ions,sequencing amplification compounds, short primers, dNTPs, enzymes,short-failed Polymerase Chain Reaction/Capillary Electrophoresisproducts, salts from Polymerase Chain Reaction/Capillary Electrophoresisproducts, or unincorporated dideoxyNTPs.
 7. The microfluidic basedsequencing reaction purification device of claim 1, wherein, after thesequencing reaction is completed, the device is configured to introduceunbound silane into the DNA sequencing sample. 8.-25. (canceled)
 26. Themicrofluidic sequencing reaction purification device of claim 7, whereinthe reagent comprises a binding buffer.
 27. The microfluidic sequencingreaction purification device of claim 26, wherein the high-surface area,convoluted material comprises frit or wool.
 28. The microfluidicsequencing reaction purification device of claim 26, wherein themicrostructures comprise microbeads, tubes, or plates.
 29. Themicrofluidic sequencing reaction purification device of claim 26,wherein the microstructures comprise polystyrene, latex, agarose, anion-exchange resin, an immobilized metal affinity chromatography resin,or any other substrate or resin capable of being coated by or bound tothe ion exchanger.
 30. The microfluidic sequencing reaction purificationdevice of claim 26, wherein the sequencing contaminants comprise salts,free salts, salt ions, ions, sequencing amplification compounds, shortprimers, dNTPs, enzymes, short-failed Polymerase ChainReaction/Capillary Electrophoresis products, salts from Polymerase ChainReaction/Capillary Electrophoresis products, or unincorporateddideoxyNTPs. 31.-32. (canceled)
 33. The microfluidic sequencing reactionpurification device of claim 7, wherein the reagent comprises ahybridization-based pull-out oligonucleotide selected to becomplementary to a sequencing contaminant.
 34. The microfluidicsequencing reaction purification device of claim 33, wherein thehigh-surface area, convoluted material comprises frit or wool.
 35. Themicrofluidic sequencing reaction purification device of claim 33,wherein the microstructures comprise microbeads, tubes, or plates. 36.The microfluidic sequencing reaction purification device of claim 33,wherein the microstructures comprise polystyrene, latex, agarose, anion-exchange resin, an immobilized metal affinity chromatography resin,or any other substrate or resin capable of being coated by or bound tothe ion exchanger.
 37. The microfluidic sequencing reaction purificationdevice of claim 33, wherein the sequencing contaminants comprise salts,free salts, salt ions, ions, sequencing amplification compounds, shortprimers, dNTPs, enzymes, short-failed Polymerase ChainReaction/Capillary Electrophoresis products, salts from Polymerase ChainReaction/Capillary Electrophoresis products, or unincorporateddideoxyNTPs.
 38. The microfluidic sequencing reaction purificationdevice of claim 33, wherein the microstructures are nonmagnetic,magnetic, or paramagnetic.
 39. The microfluidic sequencing reactionpurification device of claim 33, wherein, after the sequencing reactionis completed, the device is configured to introduce into the DNAsequencing sample unbound hybridization-based pull-out oligonucleotidesselected to be complementary to the sequencing contaminants.
 40. Themicrofluidic sequencing reaction purification device of claim 7, whereinthe reagent comprises any combination of silane, an ion exchanger, areagent adapted to sequester the sequencing contaminants based on thesize and charge of the sequencing contaminants, a binding buffer, or ahybridization-based pull-out oligonucleotide selected to becomplementary to a sequencing contaminant, as well as their unboundanalogues. 41.-48. (canceled)